Campylobacter jejuni (C. jejuni) is a food-borne pathogen that is the leading cause of human acute gastroenteritis in developed countries. Its regular hosts are live stock, in particular chicken and cattle. Infection with C. jejuni is also associated with several long-term consequences, the most severe being the autoimmune diseases Miller-Fisher syndrome and Guillain-Barré syndrome. These are evoked by antibodies of the mammalian host against the mimicry of mammalian ganglioside structures on the surface of the pathogen which then also attack the host's own gangliosides. This molecular mimicry is one of the reasons why there are currently no efficient vaccines against C. jejuni available because it excludes the use of attenuated or killed C. jejuni cells as vaccines.
US Patent 2007/065461 teaches a vaccine composed of at least one capsular polysaccharide (CPS) of C. jejuni optionally linked in vitro to a carrier protein. Injection of this conjugate into mice and apes protected against later intranasal challenge with C. jejuni. Production of this vaccine requires isolation and purification of the CPS as well as chemical linkage to the carrier protein and further purification steps.
Poly et al. (Infection and Immunity, 75:3425-3433, 2008) describe C. jejuni strains lacking the ganglioside mimicry structures that are currently tested as vaccine candidates.
Once glycosylation was considered to be specifically a eukaryotic phenomenon but was later shown to be widespread in both the Archaea and Eubacteria domains. Bacterial O- and N-linkages are formed with a wider range of sugars than those observed in eukaryotic glycoproteins. Glycosidic N-glycosylation of proteins in procaryotes was first demonstrated in C. jejuni. (Szymanski et al., Molecular Microbiology 32:1022-1030, 1999). The glycosylation machinery of C. jejuni has been characterized and has even been success-fully transferred to E. coli, where active N-glycosylation of proteins was demonstrated (Wacker et al., Science, 298:1790-1793, 2002). The gene locus of C. jejuni termed pgl (for protein glycosylation) is involved in the glycosylation of multiple proteins. Its mutational silencing results in loss of immunogenicity in multiple proteins.
US patent application 2006/0165728 A1 identifies a specific and highly immunogenic heptasaccharide that is common to at least several Campylobacter species and numerous strains that are important as human and veterinary pathogens. The heptasaccharide has the following formula (I):GalNAc-a1,4-GalNAc-a1,4-[Glc-β-1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-Bac,wherein Bac (also termed bacillosamine) is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose, GalNAc is N-Acetyl-galactosamine and Glc is glucose. This glycan moiety is a component of multiple glycoproteins. In C. jejuni the N-glycan is important for the inter-action of C. jejuni with host cells. Mutations in the glycosylation machinery lead to decreased colonisation of intestinal tracts in mice. Furthermore, pharmaceutical compositions comprising either (i) said heptasaccharide or a conjugate thereof or (ii) an antibody directed against said heptasaccharide are suggested for vaccination use in live stock, specifically in poultry.
The genus Salmonella is a member of the family Enterobacteriaceae. The genus is composed of Gram-negative bacilli that are facultative anaerobic and flagellated (motile). They possess three major antigens, the “H” or flagellar antigen, the “O” or somatic antigen (part of the LPS moiety) and the “Vi” or capsular antigen (referred to as “K” in other Enterobacteriaceae). Salmonellae also possess the LPS endotoxin characteristic of Gram-negative bacteria. LPS is composed of three domains: The lipid A part, also known as endotoxin, anchors LPS molecules in the outer membrane with its fatty acid chains. It is connected through the inner core consisting of heptoses and KDO (3-deoxy-D-manno-octulosonic acid) with the outer core containing hexoses and N-acetylhexoses. Linked to the last glucose of the outer core is the polymeric O-antigen region. This region is composed of 16 to >100 repeats of an oligosaccharide structure containing four to six monosaccharides. The endotoxic lipid A part evokes fever and can activate complement, kinin and clotting factors.
For some time Salmonella strains have been of interest for producing and presenting bacterial immunogens. For example, the genes encoding the enzymes for the biosynthesis of O-antigen of Shigella were genomically integrated into an aroA vaccination strain of Salmonella enterica serovar Typhimurium, which then produced a hybrid LPS (Fait et al., Microbial Pathogenesis 20:11-30, 1996). Also, clusters necessary for O-antigen biosynthesis of Salmonella dysenteriae were cloned into a stable expression vector, which was then transferred into the typhoid fever vaccination strain Ty21a. The resulting strain produces hybrid LPS and induces protective immunity against challenge with S. dysenteriae (DE Qui Xu et al., Vaccine 25: 6167-6175, 2007).
U.S. Pat. No. 6,399,074 B1 discloses a life attenuated Salmonella vaccine for protecting birds against infection by avian pathogenic gram-negative microbes. The vaccine is a recombinant Salmonella strain expressing the O-antigen of an avian pathogenic gram-negative microbe such as E. coli O78 that is pathogenic in poultry. The recombinant Salmonella strain does not express Salmonella O-antigen due to a mutation in the O-antigen polymerase rfz (new gene nomenclature wzy).
In view of the above prior art it is the objective of the present invention to provide an effective and safe, easily mass-produced, long-acting and cheap vaccine composition for preventing and/or treating Campylobacter infections in humans and animals, in particular in live stock, more particular in poultry.
This objective is solved by providing in a first aspect a Salmonella enterica that comprises at least one pgl operon of Campylobacter jejuni or a functional derivative thereof and presents at least one N-glycan of Campylobacter jejuni or N-glycan derivative thereof on its cell surface.
The Salmonella strain useful for the present invention can be any strain that is or can be sufficiently attenuated to allow for its non-pathological administration to humans and/or animals in life and/or dead form. Preferred Salmonella strains are Salmonella enterica strains selected from the group consisting of Salmonella Typhimurium, enteriditis, heidelberg, gallinorum, hadar, agona, kentucky, typhi and infantis, more preferably Salmonella enterica serovar Typhimurium strains. Salmonella Typhimurium is especially useful for vaccination purposes because the genome sequence is fully characterized and many animal studies confirm its safe medical use.
The term “pgl operon” as used herein refers to any physiologically active N-glycosylation cluster of C. jejuni genes capable of N-glycosylating homologous or heterologous structures produced by the Salmonella strain of the invention. The pgl operon in C. jejuni encodes all enzymes necessary for the synthesis of the C. jejuni N-glycan heptasaccharide, its transport through the inner membrane and the transfer to proteins. PglD, E, F code for the enzymes involved in bacillosamine biosynthesis, PglC transfers phosphorylated bacillosamine to undecaprenylphosphate and PglA, H and J add the GalNAc residues. The branching Glc is attached by PglI. The transfer of the completed heptasaccharide occurs through action of PglK and the oligosaccharyltransferase PglB transfers the N-glycan to protein.
A functional derivative of a pgl operon is a cluster of genes derived from any C. jejuni pgl operon having deletions, mutations and/or substitutions of nucleotide(s) or whole genes but still capable of producing a linkable oligo- or polysaccharide that can be linked to homologous or heterologous structures produced by the Salmonella strain of the invention. One or more pgl operons or derivatives thereof can be integrated into the chromosome of the Salmonella strain or it/they can be introduced as part of at least one plasmid. Chromosomal integration is preferred because it is more stable compared to plasmid vectors, the loss of which could occur during propagation. It is noted that the Salmonella strain of the invention may comprise more than one pgl operon or derivative thereof producing one or more N-glycans or derivative(s) thereof. As a matter of fact, it is preferred that the strain of the invention has more than one type of pgl operon resulting in more than one N-glycan structure, which can be of advantage for eliciting a more diverse immune response in a human or animal against different C. jejuni strains.
It is also noted that the expression level of the C. jejuni N-glycan can optionally be regulated by the use of different promoters upstream of the pgl operon, including, but not limited to, promoters of ribosomal protein genes, e.g. spc or rpsm as well as promoters from antibiotic-resistance encoding genes like bla or similar and preferably strong promoters. This type of regulation is available for plasmid-encoded or genomically integrated pgl operons. Furthermore, plasmid stability can optionally be enhanced by including essential genes on the plasmid while deleting these genes in the genome of the Salmonella strain of the invention. Preferred targets encompass for example the genes encoding the tRNA-transferases like CysS.
In a preferred embodiment, the Salmonella strain of the invention is one comprising at least one pgl operon, wherein one or more genes for bacillosamine biosynthesis are inactivated by mutation and/or partial or complete deletion, preferably by partial and/or complete deletion of the genes D, E, F, G. In a most preferred embodiment the pglE, F and G genes of the pgl operon are completely deleted and the pglD gene is partially deleted, for example the pglD open reading frame (ORF) terminates after 270 base pairs (the full length ORF contains 612 base pairs).
In a further preferred embodiment the pglB gene of the pgl operon is inactivated, meaning that the corresponding oligosaccharyltransferase B is either not expressed or at least enzymatically inactivated. The pglB gene product transfers the N-glycan to a specific polypeptide acceptor site further described below. Inactivation of the transferase leads to the N-glycan or N-glycan derivative being exclusively bound to the O-antigen acceptor lipid A core in Salmonella. 
In a most preferred embodiment the pgl derivative is one, wherein one or more genes for bacillosamine biosynthesis, pg D, E, F, G, and transfer are inactivated and the pglB gene is inactivated, too. This embodiment leads to the exchange of GlcNAc for bacillosamine resulting in increased cellular presentation as well as to transfer of the modified heptasaccharide to lipid A core instead of to polypeptide acceptors.
The at least one N-glycan of C. jejuni or N-glycan derivative thereof can be any N-glycan produced by any pgl operon of Campylobacter jejuni or a functional derivative thereof. It is of course preferred that the N-glycan is still immunogenic, i.e. elicits an immune response specific for C. jejuni. 
In a preferred embodiment, the N-glycan is the heptasaccharide of formula (I) as described above, i.e. GalNAc-a1,4-GalNAc-a1,4-[Glc-β-1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-Bac, wherein Bac (also termed bacillosamine) is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose.
The preferred pgl operon, wherein the genes for bacillosamine biosynthesis are inactivated, preferably mostly or completely deleted, leads to the synthesis of an N-glycan derivative, i.e. the heptasaccharide of formula (II), being GalNAc-a1,4-GalNAc-a1,4-[Glc-R-1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-GlcNAc.
Surprisingly, the N-glycan derivative of formula (II) is presented in higher amounts than the N-glycan of formula (I) on the cells surface of the Salmonella strains of the present invention and is also immunogenic. This is experimentally confirmed in the example section below.
In a preferred embodiment the N-glycan(s) or derivative(s) resulting from the at least one pgl operon or derivative thereof can be linked to at least one homologous or heterologous Salmonella polypeptide that will eventually be transferred to and presented on the cell surface. Preferably the at least one N-glycan or N-glycan derivative is linked to a polypeptide comprising at least one consensus sequon N-Z-S/T (see Nita-Lazar M et al., Glycobiology. 2005; 15(4):361-7), preferably D/E-X-N-Z-S/T (SEQ ID NO: 1), wherein X and Z may be any natural amino acid except Pro (see Kowarik et al. EMBO J. 2006; 25(9):1957-66).
The polypeptide linked to the N-glycan (derivative) may be any type of polypeptide such as a pure polypeptide (only amino acids) or a posttranslationally modified polypeptide, e.g. a lipid-linked polypeptide.
For heterologous polypeptides as carriers of the N-glycan(s) (derivatives) it is preferred that they comprise the signal sequence MKKILLSVLTTFVAVVLAAC (SEQ ID NO: 2) directing the N-linked conjugate to the outer membrane of the cell and wherein the LAAC motif (SEQ ID NO: 3) is used for acylation of the cysteine residue, which anchors the polypeptide in the outer membrane (see also Kowarik et al., EMBO J. May 3; 25(9):1957-66, 2006).
In the most preferred embodiment the at least one N-glycan or derivative thereof resulting from the at least one pgl operon or derivative thereof is linked to the Salmonella lipid A core or a functionally equivalent derivative thereof. The Lipid A core of Salmonella is an oligosaccharide structure consisting of hexoses, N-acetylhexoses, heptoses and KDO (3-deoxy-D-manno-octulosonic acid) linked through two glucosamines to six fatty acid chains anchoring the structure in the outer membrane of the bacterium. A functionally equivalent derivative of the lipid A core is one capable of accepting one or more glycans or derivatives thereof and presenting them on the cell surface. It is noted that in this case the N-glycan or derivative thereof is not N-linked because the Salmonella structure lipid A is not a polypeptide. The N-glycan is preferably linked to Glcll in the lipid A core or a functional derivative thereof.
Preferably the at least one N-glycan or derivative thereof takes the place of the O-antigen side chains in LPS (lipopolysaccharide). The inner and outer lipid A core of Salmonella remains unchanged while O-antigen biosynthesis is abolished through mutation of wbaP. The N-glycan is then transferred by the O-antigen ligase WaaL and linked to the Glcll residue of the lipidA outer core oligosaccharide structure.
It is preferred and for medical uses highly important that the Salmonella strain of the invention does not elicit pathogenic effects when administered to an animal or human in live and/or inactivated form. The skilled person is aware of many ways of attenuating virulent Samonella species by mutation. Preferred mutations for attenuating Salmonella strains for use in the present invention are selected from the group consisting of pab, pur, aro, aroA, asd, dap, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, phoP, phoQ, rfc, poxA and galU. One or more of these mutations may be present. Mutations aroA, cya and/or crp are more preferred.
The O-antigen biosynthesis genes of Salmonella are clustered in the rfb locus, a hypervariable DNA region of the Salmonella chromosome. Partial or full inactivation has been associated with attenuation of Salmonella strains. On the other hand, the O-antigen is also an important antigenic determinant for inducing immunity in a host.
In a particularly preferred embodiment the Salmonella strain of the present invention is attenuated by partial or full inactivation of the expression of the O-antigen, preferably by one or more mutations and/or deletions in the rfb gene cluster, more preferably in the wbaP gene, most preferably deletion of the wbaP gene.
It is understood that as used herein the terms “rfb locus” and “wbaP gene” are meant to encompass any corresponding locus and gene in any Salmonella strain that is capable of expressing O-antigen or related antigens.
The wbaP gene product is the phosphogalactosyltransferase which starts O-antigen biosynthesis by adding phosphogalactose to undecaprenylphosphate. Its inactivation/deletion leads to complete abolishment of the O-antigen synthesis, the sugar product of which competes with the N-glycan(s) (derivatives) of C. jejuni for the lipid carrier undecaprenylphosphate and for the transfer by ligase WaaL. pgl locus-induced protein N-glycosylation and wzy-dependent O-antigen synthesis in bacteria are homologous processes. It was found that the Salmonella O-antigen ligase WaaL has relaxed substrate specificity and that it can transfer C. jejuni N-glycan to Salmonella lipid A core.
Hence, in a most preferred embodiment the Salmonella strain of the invention is mutated in the wbaP gene inactivating the phosphogalactosyltransferase enzyme. It is noted that this type of O-antigen inactivation has not been described before for vaccination purposes and is superior to presently known O-antigen negative mutants, because it is genetically defined and allows for increasing the amount of C. jejuni N-glycans (derivatives) presented on the cell surface of Salmonella strains.
Therefore and as an independent invention, the present invention also relates to a Salmonella strain mutated, preferably deleted, and thus inactivated in the wbaP gene, that is useful for vaccine uses of Salmonella strains as such as well as Salmonella strains as carriers of heterologous antigens, preferably glycosylated, more preferably N-glycosylated antigens.
In a most preferred embodiment the invention is directed to Salmonella enterica, preferably a serovar typhimurium strain, that
(a) comprises
                (i) at least one pgl operon of Campylobacter jejuni or a functional derivative thereof, preferably at least one pgl operon, wherein one or more genes for bacillosamine biosynthesis are inactivated and        (ii) mutations and/or deletions in the wbaP gene leading to complete inactivation of O-antigen biosynthesis,(b) and presents at least one N-glycan of Campylobacter jejuni or N-glycan derivative thereof, preferably (I) GalNAc-a1,4-GalNAc-a1,4-[Glc-β-1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose and/or (II) GalNAc-a1,4-GalNAc-a1,4-[Glc-β-1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-GlcNAc on its cell surface.        
The above-described Salmonella strains of the invention are highly immunogenic and produce immune responses against C. jejuni infections. Furthermore, once prepared they can be easily propagated and mass-produced. As an add-on advantage the administration thereof to an animal or human provides immunity against C. jejuni and Salmonella infections. They can be administered as dead or live vaccines, live vaccines allowing for prolonged propagation and sustained immune stimulus in the host as well as full immune responses without adjuvants.
Therefore, the present invention also relates to the medical use of live or dead Salmonella strains of the present invention, in particular for preparing a medicament, preferably a vaccine.
Preferably, the medicament is useful for the prevention and/or treatment of Campylobacter jejuni and optionally Salmonella infections, preferably infections in life stock, more preferably in cattle and poultry, most preferably in poultry such as chicken, turkey, goose and ducks.
A third aspect of the present invention relates to a pharmaceutical composition, food or feed (additive) comprising dead or live Salmonella enterica of the present invention and a physiologically acceptable excipient.
For example, a pharmaceutical composition of the present invention can be prepared by medium or large scale growth of Salmonella strains of the invention containing either the at least one plasmid-encoded or chromosome-integrated pgl operon or derivative thereof. These Salmonella can be used directly or be formulated to accommodate the specific target human or animal and the specific route of administration. Pharmaceutical compositions comprising live Samonella are preferred for obvious reasons.
Alternatively, the invention relates to a food or feed for humans or animals, preferably life stock, more preferably poultry, comprising dead or live Salmonella enterica of the present invention and a physiologically acceptable excipient and/or food stuff. For example, such a feed would greatly reduce C. jejuni colonisation of poultry flocks and consequently decrease the chance of human infections by C. jejuni and also Salmonella infections through contaminated meat.
A fourth aspect of the present invention is directed to a method for treating and/or preventing C. jejuni and optionally Salmonella infections, comprising administration of a Salmonella enterica, pharmaceutical composition, food or feed of the present invention to a human or animal in need thereof in a physiologically active amount.
For therapeutic and/or prophylactic use the pharmaceutical compositions of the invention may be administered in any conventional dosage form in any conventional manner. Routes of administration include, but are not limited to, intravenously, intramuscularly, subcutaneously, intranasally, intrasynovially, by infusion, sublingually, transdermally, orally (e.g. gavage), topically or by inhalation. The preferred modes of administration are oral, intravenous and intranasal, oral and intranasal being most preferred.
The Salmonella of the invention may be administered alone or in combination with adjuvants that enhance stability and/or immunogenicity of the bacteria, facilitate administration of pharmaceutical compositions containing them, provide increased dissolution or dispersion, increase propagative activity, provide adjunct therapy, and the like, including other active ingredients.
Pharmaceutical dosage forms of the Salmonella described herein include pharmaceutically acceptable carriers and/or adjuvants known to those of ordinary skill in the art. These carriers and adjuvants include, for example, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, buffer substances, water, salts, electrolytes, cellulose-based substances, gelatine, water, pretrolatum, animal or vegetable oil, mineral or synthetic oil, saline, dextrose or other saccharide and glycol compounds such as ethylene glycol, propylene glycol or polyethylene glycol, antioxidants, lactate, etc. Preferred dosage forms include tablets, capsules, solutions, suspensions, emulsions, reconstitutable powders and transdermal patches. Methods for preparing dosage forms are well known, see, for example, H. C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th ed., Lea and Febiger (1990) and, in particular, Pastoret et al., Veterinary Vaccinology, Elsevier March 1999). Dosage levels and requirements are well-recognized in the art and may be selected by those of ordinary skill in the art from available methods and techniques suitable for a particular patient. As the skilled artisan will appreciate, lower or higher doses may be required depending on particular factors. For instance, specific doses and treatment regimens will depend on factors such as the patient's (human or animal) general health profile, the severity and course of the patient's disorder or disposition thereto, and the judgment of the treating physician or veterinarian.
In a preferred embodiment for oral vaccination the regimen consists of administration of Salmonella containing the pgl operon or derivative thereof either on plasmid or integrated into the chromosome on day 1 or 2 after hatch of the chicks with about 106 cfu (colony forming units) per chick with a boost at days 14 or 21 after hatch with the same amount of bacteria. These two administrations will provide enough stimulation for the immune system to build up a response against C. jejuni N-glycan or derivatives thereof and also against Salmonella proteins to provide protection against later colonisation of the chickens. An alternative for vaccinating chicks is by intravenous injection of inactivated, e.g. heat-inactivated or formalin-inactivated bacteria at day 1 or 2 after hatch and a boost at day 14 or 21. As a further option, chicks may also be vaccinated only once at a later time point up to 3 weeks of age, either intravenously with heat-inactivated or formalin-inactivated bacteria or intragastrically with live bacteria.
Last but not least, the present invention is about a method of producing Salmonella enterica according to the invention, comprising the step(s) of    (i) introducing into Salmonella enterica, preferably by at least one plasmid vector or by genomic integration, at least one pgl operon of C. jejuni or a functional derivative thereof, preferably at least one pgl operon, wherein one or more, preferably all genes for bacillosamine biosynthesis are inactivated, and    (ii) preferably introducing mutations and/or deletions in the wbaP gene leading to complete inactivation of O-antigen biosynthesis.
In the following the present invention will be further illustrated with reference to specific embodiments and experiments which are not intended to be interpreted as limiting the scope of the invention as presented by the appended claims.